The glucocorticoid hormone regulatory sequences in the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) have been localized between 100 and 200 nucleotides upstream from the cap site in the LTR. Our earlier experiments suggested that these sequences conferred a negative effect on transcription in the absence of hormone. Other experiments show that nucleosomes are phased in the vicinity of the hormone regulatory sites, indicating that the region is highly organized at the chromatin level. These results predict that deletion mutagenesis would have multiple effects, perturbing both binding sites for regulatory factors and the underlying nucleoprotein structure. We have developed a new method, "oligo-scanning mutagenesis," that permits high resolution probing of DNA-protein interactions in the region without major alteration in the overall structure. This technique permits the oligonucleotide-directed introduction of alterations in DNA sequence as large as 10 nucleotides in a one-step procedure. The method is precise (recovered mutants contain only the predicted alteration) and efficient (predicted mutants routinely represent 3-10% of recovered clones). A series of mutations induced with this technique confirm the earlier results obtained with deletion mutagenesis. That is, mutant combinations that significantly perturb the hormone response region lead to an increased constitutive level of transcription in the absence of hormone. These results indicate the hormone response is complex, involving both negative effects minus activated receptor and positive effects in the presence of hormone.